Arterial stiffness plays a significant role in the development and progression of adverse cardiovascular events and all-cause mortality. This observational study aims to explore the relationship between six acute phase proteins namely, ceruloplasmin, alpha-1-antitrypsin, orosomucoid, haptoglobin, complement C3 and C-reactive protein (CRP), and carotid-femoral pulse wave velocity (c-f PWV) in a population-based cohort, and to also explore the effect of low-grade inflammation on the relationship between diabetes and c-f PWV.The study consisted of participants from the Malmö Diet and Cancer study with data from baseline examinations (1991–1994) and follow-up examinations (2007–2012). Arterial stiffness was measured at follow-up by determining c-f PWV. After excluding participants with missing data, the total study population included 2338 subjects. General linear models were used to assess the relationship between baseline acute phase proteins and c-f PWV.th quartile vs 1st quartile of alpha-1-antitrypsin (geometric mean: 10.32 m/s vs 10.04 m/s) (p<0.05), C3 (10.35 m/s vs 10.06 m/s) (p<0.05) and CRP (10.37 m/s vs 9.96 m/s) (p<0.001) showed significant association with c-f PWV. Diabetes at follow-up was also associated with high c-f PWV, however, this relationship was independent of low grade inflammation.After adjusting for traditional risk factors the participants in the 4Alpha-1-antitrypsin, C3 and CRP are associated with arterial stiffness. The results indicate that low grade inflammation is associated with arterial stiffness in addition to established cardiovascular risk factors. Arterial stiffness is an important risk determinant for adverse cardiovascular events and all-cause mortality , 2. VariThere are multiple risk factors that affect arterial stiffness. Age and blood pressure have been shown to be associated with arterial stiffness , 12. AddThere is evidence supporting the fact that inflammation is associated with adverse cardiovascular events, as shown by previous studies –19. It hA recent cross-sectional study from the Malmö Diet and Cancer study (MDC) reported that waist circumference, raised fasting glucose, insulin resistance and diabetes, and dyslipidemia were associated with increased c-f PWV . WhetherThe aim of this observational study is to investigate the association between six acute phase proteins namely, ceruloplasmin, alpha-1-antitrypsin, orosomucoid, haptoglobin, complement C3 and CRP on the one hand and c-f PWV on the other hand in a population-based cohort, followed up for 17 years. Another aim was to study whether the relationship between diabetes and c-f PWV is modified by low-grade inflammation.The Malmö Diet and Cancer (MDC) study is a large prospective cohort, compromising of men and women from the city of Malmö, Sweden . A randoThe study conforms to the ethical guidelines of the 1975 Declaration of Helsinki. It was approved by the Lund University Ethical Committee (LU 51–90 and LU 532–2006). All the participants provided written informed consent.Data from baseline examination was collected by means of a self-administered questionnaire, physical examination and collection of blood samples . InformaDiabetes was defined based on a self-reported physician’s diagnosis of diabetes, use of anti-diabetic medication, or fasting whole blood glucose ≥ 6.1 mmol/L (corresponding to a plasma glucose of 7.0 mmol/L) . Informa® CRP latex assay  on an ADVIA®1650 Chemistry System .The plasma levels of ceruloplasmin, orosomucoid, haptoglobin, alpha-1-antitrypsin and C3 were analyzed using Cobas c-systems  and have been explained elsewhere . High-seThe follow-up examinations were carried out between 2007 and 2012 and comprised of a self-administered questionnaire, blood samples collection and physical examinations. Blood pressure was measured after 5 min rest in supine position using the OMRON M5-1 IntelliSense device. Fasting blood samples were collected for measurement of plasma glucose (p-glucose) using HemoCue . Oral glucose tolerance test (OGTT) was conducted following an overnight fast after intake of 75 g of glucose and plasma glucose measurements were done before intake and after 120 min. Fasting plasma glucose of at least 7.0 mmol/L or 2 hour glucose of at least 11.0 mmol/L, verified subsequently by a fasting plasma glucose, was diagnosed as diabetes.Diabetic cases at follow-up were defined on the basis of six different national and regional diabetes registers. These have been described in detail elsewhere . AdditioThe measurements for the c-f PWV were carried out on average 261 days after the first visit in the follow-up examination. They were conducted by using applanation tonometry  following a specific study protocol. The measurements were done with the patients in supine position after 5 minutes resting. Pulse curves from the carotid and femoral arteries were obtained with a pressure sensitive probe. For the purpose of calculation, the distance was measured from the suprasternal notch to the umbilicus and the umbilicus to the measuring point at the femoral artery subtracting the suprasternal notch to the measuring point at the carotid artery. ECG was registered simultaneously and the time from the peak of the R-wave on ECG to the foot of the pulse wave at the carotid and femoral arteries was automatically calculated. Each participant had varying number of successful measurements, ranging between one and five. The aim was to obtain three measurements each, which was possible in 86.7% of the cases. Mean c-f PWV was determined from these measurements . Heart rInter-observer variability was determined twice by two technicians in 17 and 13 participants, respectively. Inter-observer difference was 5.0% (± SD4.0) and 7.2% (±SD9.9), respectively.th -75th percentiles) or percentages. The 2007 European Society of Hypertension-European society of Cardiology (ESH-ESC) guidelines for the management of hypertension proposed a cut off value of c-f PWV of 12 m/s [All statistical analyses were performed using IBM SPSS Statistics version 22 . Variables such as c-f PWV, ceruloplasmin, C3 and CRP were natural log (ln) transformed due to their skewed distribution. Sex-specific quartiles of the acute phase proteins were created, i.e., four quartiles with the same proportion of men and women in each quartile. The baseline and follow-up characteristics of the study population were presented as means ± standard deviation (SD), median  (p<0.05), C3 (10.35 m/s vs 10.06 m/s) (p<0.05) and CRP (10.37 m/s vs 9.96 m/s) (p<0.001). The p-values for trend across quartiles of acute phase proteins are shown in When the different quartiles of the acute phase proteins were compared, the association between all the acute phase proteins and c-f PWV were significant for the participants in the 4p<0.001). After adjusting for the acute phase proteins and other covariates the c-f PWV remained significantly higher for individuals with diabetes vs those without diabetes (geometric means: 10.61 m/s vs 10.14 m/s) (p<0.001).C-f PWV was also compared in individuals with and without diabetes at the follow-up examination. At the re-examination, 305 individuals had diabetes and 2033 were non-diabetic. As expected, diabetes was significantly associated with c-f PWV (geometric means: 11.16 m/s vs 10.06 m/s)  and 27 had prevalent cardiovascular disease  at baseline. At re-examination, the number of participants with rheumatic and cardiovascular disease was 88 and 171 respectively.Regarding anti-inflammatory medication, 0.3% (n = 7) and 3.5% (n = 82) of the subjects were taking steroids and aspirin respectively at baseline. These were also excluded for the purpose of the sensitivity analysis. The results showed that the association was still significant between alpha-1-antitrypsin, C3, CRP and arterial stiffness.The results from this large, observational, population-based study show that acute phase proteins alpha-1-antitrypsin, C3 and CRP are associated with future c-f PWV, even after controlling for traditional cardiovascular risk factors. This suggests that low grade inflammation may play a role in the pathogenesis of arterial stiffening, hence contributing to increased cardiovascular risk. Since inflammation is associated with increased incidence of diabetes, we also hypothesized that inflammation could modify the relationship between diabetes and arterial stiffness. We could confirm that diabetes at follow-up was strongly associated with c-f PWV, however, this relationship was independent of baseline low-grade inflammation in this study.The mechanism as to how these inflammatory biomarkers modify arterial stiffness is unclear. Numerous studies have looked at the link between inflammation and arterial stiffness, but these have consisted mostly of study populations with chronic inflammatory diseases –24. It set al. demonstrated that a group of patients with alpha-1–antitrypsin deficiency and related chronic obstructive pulmonary disease had increased aortic stiffness [SERPINA1) gene, a study undertaken by Dahl et al. concluded that alpha-1-antitrypsin deficiency in ZZ and MZ genotypes is associated with reduced blood pressure in individuals with ischemic heart disease (IHD) as compared to MM/MS genotypes. Additionally, MZ heterozygosity was associated with reduced risk of ischemic cerebrovascular and IHD [Alpha-1-antitrypsin is a proteinase inhibitor with the primary function of inhibiting proteolytic enzymes like neutrophil elastase. Hence, it plays a role in maintaining the vascular structure. Our results show that there is association between alpha-1-antitrypsin and arterial stiffness after adjustment for the traditional risk factors. This is an interesting finding as previous studies have assessed the relationship between alpha-1-antitrypsin and various cardiovascular diseases with conflicting results , 37. Mostiffness . Looking and IHD , the proat al. proposed a mechanism to explain the association between C3 and arterial stiffness [It has also been observed in the present study that C3 is associated with c-f PWV. To our knowledge only one previous study has looked at the association between C3 and c-f PWV. The study included middle aged women with Systemic Lupus Erythematosus (SLE) and demonstrated positive association between C3 and CRP on the one hand with aortic stiffness on the other . This istiffness . Their set al. demonstrated that there is an association between CRP and arterial stiffness in older adults [et al. showed that although CRP and peripheral pulse pressure were associated, this was not seen with aortic stiffness [et al. explored the association between circulating CRP levels, the CRP genotype and PWV. Interestingly, the study concluded that although there exists an association between circulating levels of CRP and c-f PWV, there is no causal relationship between CRP and arterial stiffness [The results from the present study show that CRP levels and c-f PWV are associated. These findings are supported by studies that have shown association of CRP with arterial stiffness in apparently healthy individuals , 43. Stir adults . Resultsr adults , 46. In tiffness . It has tiffness . Howevertiffness .There is substantial evidence suggesting association between diabetes and arterial stiffness , 50, 51.One of the strengths of this study is that it is a large population-based cohort with a long follow-up period. Although it can be argued that the final study population was health-selected as many individuals with poor health died before the re-examination, it has been demonstrated that the study cohort was representative of the population in terms of baseline characteristics, when examined through a mailed health survey (participation rate 75%) . AnotherEven though c-f PWV is a valid measure of the arterial stiffness in the aortic and aorto-iliac vascular tree, it should be acknowledged that c-f PWV does not reflect the heterogeneity which occurs in various primary and secondary branches. A recent study by Weir-McCall et al. demonstrated differences in carotid-femoral measured PWV and MRI measured aortic PWV, which can be for the most part be explained by use of simple surface measurements . NeverthIn conclusion, this prospective population-based cohort study shows that alpha-1-antitrypsin, C3 and CRP are associated with c-f PWV and, therefore, arterial stiffness. This observation suggests that inflammation may be involved in the pathogenesis of arterial stiffening. The results also indicated that individuals with diabetes had increased c-f PWV. However, this relationship was not explained by raised inflammatory proteins. A better understanding of the underlying mechanism of association between low grade inflammation and arterial stiffness is necessary, due to its important implications on future diagnosis, risk stratification, and potential therapeutic approach of cardiovascular diseases.


Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection. Feline herpesvirus 1 (FHV-1), an enveloped double-stranded DNA virus, belongs to the Varicellovirus genus of the subfamily Alphaherpesvirinae -THF-[BHQ-dT]GAAACTGATGTTGTCGGTGG-3’. Primers and the probe were synthesized by Takara Bio Inc.To date, specific programs for RPA primer design are not available. For this study, primers for the RPA assay were designed using the Primer 3.0 program following the “primer design considerations” for RPA . Other components included 420 nM each RPA primer, 120 nM exo probe, 14 mM magnesium acetate, and 1 μL of nucleic acid. All reagents except for the template and magnesium acetate were prepared in a master mix, which was distributed into each 0.2 ml reaction tube containing a dried enzyme pellet. One μL of nucleic acid template was added to the tubes. Subsequently, magnesium acetate was pipetted into the tube lids, the lids were closed carefully, and magnesium acetate was centrifuged into the rehydrated material using a minispin centrifuge. The sample was vortexed briefly and spun down. The tubes were immediately placed in the Genie III Instrument  and incubated at 39°C for 20 min. The fluorescence signal was monitored in real-time. A cutoff value of 3000  as follows: forward primer, 5’-GGACAGCATAAAAGCGATTG-3’; reverse primer, 5’-AACGTGAACAACGACGCAG-3’; and the probe, 5’-FAM-AATTCCAGCCCGGAGCCTCAAT-BHQ1-3’. Primers and the probe were synthesized by Takara Bio Inc. Real time PCR was performed using the Premix Ex Taq Master Mix (Takara Bio Inc.) according to the manufacturer’s instruction. The reaction was completed on the ABI 7500 Real-Time PCR System in following steps, 95°C for 3 min and then 40 cycles of 95°C for 15 s and 60°C for 30 s. The fluorescent dye FAM was used as a reporter dye in real time PCR. The dye can incorporate into the amplified product, and the fluorescent intensity is proportional with the relative amount of the PCR product, which can be measured in real time and is reflected by the Y-axis value, so called delta Rn value, on a real time PCR plot. The Rn value stands for normalized reporter value. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. A cutoff Delta Rn value of 2.000e + 005  were statistically analyzed and the co-efficient of variations was calculated to determine the reproducibility of the assay.To evaluate the sensitivity of the RPA assay, plasmid pMD19-T-TK DNA was 10-fold serially diluted to achieve DNA concentrations ranging from 10 005 see  after 40From 2013 to 2015, 120 clinical samples, i.e., nasal and ocular conjunctival swabs, were collected from cats with conjunctivitis, rhinitis or symptoms of upper respiratory tract infections and sent to our laboratory for test. They were from different veterinary clinics and two breeding catteries in Hebei Province, China. The swab was placed in 200 μL sterile phosphate-buffered saline. After centrifugation at 1000×g for 10 min at 4°C, the supernatant was collected and applied for viral DNA extraction using the TIANamp DNA Extraction Kit. DNA from each sample was finally eluted in 20 μL of nuclease free water and stored at a -80°C freezer until assays were performed .feline calicivirus and Chlamydia [feline calicivirus, Chlamydia psittaci and other viruses commonly seen in cats, i.e., FPV, BHV-1, PRV, were all tested negative  was demonstrated in Different copy numbers of pMD19-T-TK plasmid DNA were assayed by exo-RPA and real-time PCR. As shown in reaction . Thus, i3 copies of FHV-1 genomic DNA could be detected. Weigler et al developed a real time PCR method for FHV-1 detection, which had a detection limit of 240 copies of FHV-1 DNA [http://www.twistdx.co.uk/images/uploads/docs/TA01cmanual_Combined_Manual_RevI.pdf), eliminating the need of complex shipment and storage containers, which is convenient and fitting for field performance of rapid point-of-care diagnostic tests. Lastly, it has been shown that RPA primers and probes carrying 5–9 base pair mismatches do not influence the performance of RPA reactions [Reubel et al described a conventional PCR assay for FHV-1 detection . They usHV-1 DNA , in lineeactions , 19, whieactions .With the development of portable devices specifically designed for out-door use, e.g., Genie III Instrument, which are light and incorporate a rechargeable battery that can support operation for a complete day, the RPA assay developed in this study could become a useful tool for field diagnostic tests of FHV-1 infection. To our knowledge, this is the first report on the development of an RPA assay for the detection of FHV-1. In conclusion, the RPA assay is a simple, fast and cost-effective alternative to real time PCR for specific detection of FHV-1.


Dentinal canal walls are in direct contact with endodontic sealers prior to post space preparation and luting cements after post space preparation. This direct contact may affect the bond strength of intraradicular posts to root dentin. This study aimed to assess the effect of three different sealers on the bond strength of fiber posts to root dentin. n=14) according to the type of sealer used in conjunction with gutta-percha: group 1 (control) without any sealer; group 2 with AH-Plus sealer (resin based); group 3 with Dorifill sealer (ZOE-based); and group 4 with BC Sealer . Nine mm-deep post space was prepared in the canal of each specimen. Intraradicular fiber posts were cemented using dual-cure resin cement (Panavia F2.0). Sections of 1 mm thickness were made at the coronal, middle and apical thirds of the post space of each specimen. The push-out bond strength of post to root dentin was measured in a universal testing machine. The data were analyzed using one-way ANOVA and post-hoc Tukey’s test. The canals of 56 extracted single-rooted human premolars after selection and decoronation were prepared. For obturation of the canals, specimens were randomly divided into four groups (P=0.004). This value was significantly lower in BC Sealer group than AH-Plus (P=0.000) and control group (P=0.03). In middle and apical thirds, the mean push-out bond strength was not significantly different among the four groups . The mean push-out bond strength was significantly lower in apical than coronal third in AH-Plus group (P=0.001).  The mean push-out bond strength in the coronal third was significantly lower in Dorifill group compared to AH-Plus ( Application of BC Sealer and Dorifill decreased the mean push-out bond strength of intracanal post to root dentin in the coronal third in comparison to AH-Plus. Generally, gutta-percha in conjunction with a sealer is routinely used as filling materials for root canal treatment , 2. SealAfter completion of root canal treatment, proper restoration of tooth is required to restore its function and esthetics . Most enBased on previous studies, success of endodontically treated teeth with intracanal posts depends on proper selection of filling material, sealer and type of intracanal post , 11. ManA previous study has shown that zinc oxide-eugenol-based sealers decrease the retention of fiber post . The draA total of 56 single-rooted and single canal human premolar teeth, extracted due to periodontal problems, were collected for this study. Radiographs were taken to ensure the presence of a single canal. For disinfection, the teeth were immersed in 0.5% chloramine-T solution for one week. External surfaces of teeth were cleaned from debris and necrotic tissue using an ultrasonic scaler . The teeth were evaluated under a light microscope  under 10× magnification to exclude teeth with cracks. Tooth crowns were cut at the cementoenamel junction using a diamond disc and low speed handpiece under water coolant so that 13±1 mm of root length remained. Working length was determined by subtracting 1 mm from the length of a #10 K-file  after observing its tip at the apical foramen. Flaring the coronal third of the root canal was performed using #2 to 4 Gates Glidden drills . All root canals were prepared up to #45 K-file. Root canals were irrigated using 2.5% NaOCl solution after using each file. After root canal preparation, smear layer was removed using 1 mL of 17% ethylenediaminetetraacetic acid (EDTA)  for one min, 3 mL of 5.25% NaOCl and a final rinse with 5 mL of distilled water.The root canals were then dried with paper points . According to the sealer used for filling of the root canal system, the roots were randomly divided into four groups of 14 as follows: Group 1 (Control), gutta-percha without sealer; group 2, gutta-percha and AH-Plus sealer ; group 3, gutta-percha and Dorifill sealer  and group 4, Gutta-percha and BC Sealer . Root canal sealers were prepared as recommended by the manufacturers and root canals were filled using cold lateral compaction technique. Then the orifice of the root canals were sealed with temporary filling material . °C for one week. Then, 9 mm depth post space was prepared using a #2 peeso reamer  and a #2 drill  in each root canal. The canals were then rinsed with copious water and dried with paper points. Fiber posts were cemented into the root with Panavia F2.0 resin cement  according to the manufacturer’s instructions. Fiber posts were placed deep into the canal using finger pressure and resin cement was polymerized for 20 sec using VALO light curing unit . The roots were then incubated in 100% humidity at 37°C for 24 h. In order to cut cross-sectional slices, the roots were mounted in blocks containing clear polyester resin. After polymerization, dentin discs were sectioned with 1 mm thickness at the coronal, middle and apical thirds of the created post space using a Mecatome with diamond disc  under copious water irrigation. The push-out bond strength was measured in a universal testing machine  with a crosshead speed of 0.5 mm/min . Using the following formula push-out bond strength was calculated: Maximum load (N)/area of fiber post (mm2). The area of fiber post was calculated using π(R+r)[(h2+(R-r)2]/2 where R(mm), r(mm) and h(mm) are larger radius, smaller radius and the thickness of the root section, respectively. The push-out bond strength data were converted from Newtons to Megapascals (MPa). Mode of bond failure was determined under a stereomicroscope  under 10× magnification. The percentage of each mode of failure in each group was calculated. The teeth were incubated in 100% humidity at 37Data were analyzed using one-way ANOVA and post-hoc Tukey’s test. The mode of failure was classified into four types: 1) adhesive between the post and resin cement, 2) mixed with resin cement covering 0-50% of the post diameter, 3) mixed with resin cement covering 50-100% of post diameter and 4) cohesive in dentin.P=0.001), in the coronal third, pairwise comparisons were carried out and revealed that the mean push-out bond strength of fiber post to root dentin was significantly lower where Dorifill was used compared to AH-Plus (P=0.004). Also, the mean push-out bond strength of fiber post to root dentin in BC Sealer group was significantly lower than that of AH-Plus group (P=0.000) and that in control group (P=0.03). In the middle and apical third region, the mean push-out bond strength of post to root dentin was not significantly different among the four groups (P=0.407) and (P=0.065), respectively. P=0.001). However, the mean push-out bond strength was not significantly different in the coronal, middle or apical thirds in the Dorifill (P=0.321), BC Sealer (P=0.358) and control (P=0.321) groups.In AH-Plus group, the mean push-out bond strength in apical was significantly lower than coronal third  when ZOE-based sealers are used. This is probably due to the removal of a thicker layer of dentin affected by sealers and increased surface area for resin cement bond . Becauseet al. [After post space preparation, sealers or eugenol remaining on root canal dentinal walls must be eliminated in order not to decrease or prevent the polymerization reactions of luting cements . Cohen eet al.  showed tet al. .et al. [Cecchin et al.  stated tet al. -35. Moreet al. , 37, whiet al.  or BC seet al. , 40. Difet al. .et al. [et al. [In present study in AH-Plus sealer group the mean push-out bond strength was significantly lower in the apical thirds than coronal third, in agreement to the results of Gundogar et al. , with DT [et al. .et al. [This study has shown that in Dorifill group, the mean push-out bond strength in the coronal, middle and apical thirds was not significantly different, which was in agreement with the results of previous studies , 25. In et al. , 42.Considering the fact that no previous study has assessed the bond strength of fiber post to root dentin with the use of BC Sealer, the current results were compared with those on MTA-Fillapex , 43, 44 Within the limitation of this study, application of BC Sealer and Dorifill decreased the mean bond strength of fiber post to root dentin compared to AH-Plus sealer in the coronal third. This effect was not seen in the middle and apical thirds.


This VLP was designed to elicit neutralizing antibodies, to induce better T-cell responses and to activate the innate immune system. Overall, 5 groups of 10 mice were electroporated with the following constructs: pVLP-LTR-GagPro [full], pVLP-GagPro [VLP wihout RNA], pVLP-LTR-Gag [VLP immature], pVLP-Gag and pVLP-EnvBG505 [regular DNA vaccine] and a mock group. We performed ICS on the mouse spleens and performed ELISA for ENV antibodies and a Luminex assay for inflammatory cytokines. The VLP showed good binding to the neutralizing antibodies. The percentage of CD4 cells producing cytokines was 0.1% [IFNg], 0.15%[IL-2] and 0.2% [TNFa] for the construct pVLP-LTR-GagPro. The percentage of CD8 cells producing cytokines was 0.3%[IFNg], 0.2%[IL-2] and 0.25%[TNFa]. All pVLP constructs induced more antibodies for EnvBG505 than the regular DNA vaccine Env. The pVLP-LTR-GagPro induced more IL-1B than the other constructs 24 hours post-vaccination.We developed a DNA vaccine that induces the formation of a VLP  It has been more than 25 years since HIV-1 was identified as the causative agent for AIDS . The maiAs mentioned above, T cell responses are desired to be elicited by an effective HIV vaccine . It is wTraditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. As the VLP formed by DNA vaccination is similar to the HIV virus itself, we think this vaccine can work as a live vaccine, but inactivated. In other words, the vaccine will be non-replicative. Actually, will have one cycle of replication. Once the VLP is formed, it will infect a cell and its RNA will be translated into new proteins that will form a new VLP which will not contain RNA. This will result in the augmented expression of vaccine antigens in vivo. Thus this VLP of HIV can use the host-cell protein processing pathways that lead to antigen presentation via the major histocompatibility complex class I and consequent cytotoxic T-cell stimulation .in vivo assembly and budding of a mature Gag VLP containing BG505 Env and also carrying a portion of the HIV-1 genome. The idea is not to use VLP, but rather the DNA itself, as vaccine to drive the assembly and budding of the VLP in vivo. One promoter drives the expression of HIV-1 LTR-GagPro composed of HIV nucleic acid sequences, which can encode mature Gag proteins, while the LTR-GapPro RNA will be encapsulated in the particles due the presence of the Psi sequence in the LTR. This viral RNA in the VLP can trigger the innate immune system through ligation to TLR-7 and RIG-1. The other promoter expresses Rev and Env BG505. Rev protein allows correct HIV RNA processing and packaging, and Env will be incorporated into the budding VLP. The membrane of a virus, here a VLP, is the ideal place for the right conformation of the HIV envelope. It is the place where envelope exposes its CD4 binding sites, essential for neutralizing antibodies to bind. We tested this new DNA vaccine approach in mice and produced good immunogenicity results. The vaccine induced high levels of T cell responses, good titers of Env specific antibodies and was able to induce IL-1β 24 hours post-vaccination, suggesting innate immune system activation.Based on the above facts, we propose the development of a new HIV-1 DNA vaccine candidate, which is designed to elicit neutralizing antibodies [NAb] and T-cell responses concomitant with HIV-1 specific innate responses. Our approach is to develop a dual promoter DNA vaccine vector. The rationale is to have the DNA vaccine driving All genes orders were made to GeneArt ® and were human optimized. The LTRGagPro was synthetized as the first 1925 nucleotides of the prototype HIV-1 HXB2 genome. The Rev2AEnvBG505 was ordered as the Rev gene followed by a furin site (RKRR), the 2A sequence of Tomato aspermy virus (TAV) and the Env BG505 gene . The natWe built the pCMV-dualpromoter vector as a bi-cistronic eukaryotic expression vector with 2 CMV promoters and kanamycin resistance, to be a DNA vaccine vector. We cloned the LTRGagPro sequence in the BamH1 and EcoRV sites of the MCS1 and cloned the Rev2AEnvBG505 in the EcoRI and XbaI sites of the MCS2. We called the plasmid formed pVLP-LTRGagPro. The others construct, pVLP-GagPro, pVLP-LTRGag, pCMV-Gag and pCMV-Env, were built by PCR amplification of the target region and cloning of the MCS1 . Rev2aEnTo evaluate the processing of Gag proteins in the VLP, the LTR-GagPro [processed Gag] and LTR-Gag [non-processed Gag] plasmids were transfected to 293-T cells with 1.0 μg of DNA in 12 a well plate, using the reagent Lipofectamine® 2000 . 48 hours after the initiation of transfection, cells were removed and lysed in the presence of a low salinity buffer  at 4°C for 30 minutes. The lysate was clarified by centrifugation at 13,000 g. The supernatant was collected, filtered  and concentrated in a 20% sucrose buffer in an ultracentrifuge (44000 g / 45 minutes / 4°C). Then the supernatant was discarded and the VLP was resuspended in 100 μL of PBS.Proteins were separated by 12.5% SDS-PAGE and then transferred to nitrocellulose membranes  using the BIORAD transfer mini-vessel. The transfer was carried out in transfer buffer  for 16 hours at 20 Volts. After transfer, the membranes were incubated at room temperature for 2 hours in blocking buffer . Then, the primary antibody  was added and incubated for 2 hours at room temperature. At the end of this period, the membranes were washed 3 times with 1X PBS containing 0.2% Tween-20. The secondary antibody was added in the same buffer (anti-mouse immunoglobulins) and incubated for 1 hour at room temperature.Finally, the membranes were washed 5 times with PBS-Tween and developed by chemiluminescence using the reagent kit for ECL protein detection  according to the manufacturer's guidelines.Pull-down, ELISA and PCR: We transfected the pVLP-LTRGagPro into 293-T cells and collected the supernatant. Then we labelled the VLP in the supernatant with biotinylated  neutralizing antibodies . Then we pulled down the labelled VLP with Anti-Biotin Microbeads (Miltenyi).We performed an HIV-1 p24 ELISA  with the purified VLP. Additionally, we extracted the viral RNA of the purified VLP with the QIAamp Viral RNA Mini Kit  and made cDNA with the High Capacity cDNA kit . We then performed PCR for the Gag region.2 inhalation or Euthasol (100mg/kg) administered IP and a terminal blood collection via cardiac puncture was performed. The condition of the animals was monitored on daily basis. No animals showed signs of illness post treatment. And no animal died (without euthanasia) due to the experimental procedures. An IACUC protocol (IBC-13-012) was issued, for this study at the State University of New York Downstate Medical Center, where IAVI houses the animal incubators. Institutional Care and Use Committee (IACUC) specifically approved this study.We tested this vaccine in mice, and 10 animals were used for each group of vaccine tested. Balb/c animals were used. The groups were pVLP-LTRGagPro, pVLP-GagPro, pVLP-LTRGag, pCMV-Gag + pCMV-Env, and an empty vector. The animals were vaccinated by electroporation  with 20 μg of DNA, twice, every 3 weeks. Serum was collected 24 hours after the second exposure and during week 5. On week 9, animals were bled for serum then euthanized, and the spleens were removed for T cell analysis. The mice will be euthanized via COSerial dilutions of heat inactivated sera from week 5 and 9 were incubated for 1 hr on 96-well plates which were previously coated with Env BG505 protein [homemade produced], then blocked for 1 hour with 3% BSA/PBS. Plates were washed in 0.05% PBS/Tween and incubated for 1 hr with the alkaline 236 phosphatase-conjugated secondary antibodies. After washing, plates were incubated for 30 min with nitrophenylphosphate and optical density was measured at 450 nm using SoftMAx Pro GxP v5 . All data shown have the ‘no sera’ background signal subtracted.2. Cells were washed and stained with LIVE/DEAD -Aqua Viability fixable cell stain , then washed and stained for extracellular markers with CD3-Pacific Blue, CD4-PerCP Cy5.5, and CD95-APC . Cells were permeabilized with Fix Perm and stained for markers and intracellular cytokines with CD69-Texas Red, CD8-APC-H7, IL-2- PE, IFN-γ-Alexa 700, and TNF-α-PE-Cy7 . Cells were collected on an LSR-II  and analyzed using a FlowJo device . All of the data shown have the ‘Mock’ stimulated background responses subtracted.PBMC between 600,000 and 1,000,000 were incubated with gag protein and HIV-1 Consensus B Gag peptide pools  or gp 120 protein for 9 hours with brefeldin A , CD49d, and CD28  at 37°C with 5% COInflamatory cytokines were analyzed in the animal’s sera, collected 24 hr post-vaccination. We used The Inflammatory Cytokine Mouse Magnetic 4-Plex Panel for the Luminex™ , which detects GM-CSF, IL-1β, IL-6, and TNF-α. The reads were performed using a Bio-Plex 200 device . Three independent experiments were performed.A one-way ANOVA was used to compare all 5 groups in the experiments. A Tukey-Kramer multiple comparison test was used post-test. Graphics were produced using GraphPad Prism 6.0, and we performed the statistical analyses on GraphPad InStat 3.10.in vitro. The rationale was to have the DNA vaccine driving in vivo assembly and budding of a mature Gag VLPs containing BG505 Env, that also carries a portion of the HIV-1 genome. One promoter drives the expression of HIV-1 LTR-GagPro composed of native HIV nucleic acid sequences, which encodes for mature Gag proteins. The RNAm of LTR-GagPro, which will be encapsulated in the particle through Psi sequence in the LTR, can form secondary structures that induce the innate arm of the immune system. The other promoter expresses Rev and Env BG505. Rev protein allows correct HIV RNA processing and packaging . pVLP-GagPro produced a higher titer, approximately 2500 serum dilution. pVLP-LTRGag induced a serum dilution of approximately 2000 and pCMV-Gag and–Env, induced a serum dilution of approximately 1000 .In the ICS experiment we lost the samples from week 5 due a laboratory accident. Thus, we could only analyze the samples from week 9. From this experiment, we could see that pVLP-LTRGagPro induced the highest levels of cytokine production when stimulated with gag pool peptides. For CD4 T-cells, it induced 0.1, 0.2, and 0.35% of IFNγ, IL-2, and TNFα, respectively . For CD8 T-cells, it produced 0.5, 0.2 and 0.55% of IFNγ, IL-2, and TNFα, respectively  . When coWe measured the innate immune response 24 hours after the last vaccine shot. We analyzed the production of the cytokines GM-CSF, IL-1β, IL-6, and TNF-α. For GM-CSF, IL-6, and TNF-α we did not observe differences [data not shown]. We did observe an increase in the production of IL-1β for pVLP-LTRGagPro that was statistically significant, with p<0.001 .We observed that pVLP-LTRGagPro built a mature particle, with the processed Gag proteins, as shown in the Western blot experiment. pVLP-LTRGag did not show a mature Gag protein, as expected.In the pull-down experiment, we could see that the VLP (pVLP-LTRGagPro) binds well to all neutralizing antibodies used . This proved that the VLP was well assembled. Because we detected the capsid (p24) by ELISA, we determined that the BG505 envelope is expressed in the right conformation, thus exposing the CD4 binding domains, allowing it to bind to the NAbs. Cale et al, also used VLP to stabilize the env trimer and identify HIV-1-neutralizing N90-VRC38 Ab lineage–a good neutralizing antibody . These rAmong the cell types analyzed [CD4 e CD8], the pVLP-LTRGagPro showed the highest levels of all produced cytokines  when pulsed with gag peptides. This construction induced more cytokines than the regular DNA vaccine pCMV-Gag + pCMV-Env. It appears that the response was shifted to a more Th1 pattern. We think that this effect was caused because of the presence of of viral RNA that acted as an adjuvant, triggering the innate immune system. The T-cell responses against Env gp120 was low. This is desirable in a HIV vaccine, as the T-cell response to Env is associated with increasing HIV viremia . The conHigh magnitude, polyfunctional CD8 T-cell responses to Gag have been shown to be an important component of the control of viremia in HIV positive long term non-progressors and elite controllers. Therefore, Gag is considered to be an important component of HIV vaccines because a strong T cell response could result in the early clearance of HIV-infected cells via CD8 T-cells at the site of infection, control of the spread from the entry portal, and control of viremia .et al . As . As 24].We tested the innate immune responses 24 hours after vaccination. This was not ideal, and it should have been tested 4–6 hours post vaccination. Nevertheless, we were able to see a high level of IL-1β upon pVLP-LTRGagPro vaccination, which indicates the activation of the innate immune system.These results illustrate that VLPs with GagPro provide a safe and effective means of enhancing neutralizing humoral responses to particles containing gp120 proteins. Therefore, the antigenicity of these VLPs represent an attractive approach in the design of vaccines for which specific stimulation of neutralizing antibodies and cytotoxic effector functions to complex glycoproteins is desired .The vaccine tested here, pVLP-LTRGagPro, showed better immunogenic results than its controls. It showed higher ICS results for both CD4 and CD8 t-cells. The high Gag response of the T-cell and the low Env T-cell response is desirable for an HIV vaccine . This vaThe next step for this vaccine should be testing in NHP [non-human primates]. In the NHP setting, we could evaluate non-innate immune response and determine whether this vaccine is able to protect animals from infection upon challenge from an SHIV model.


P = 0.004), dominant genetic model , recessive genetic model , and allele comparison . Stratification analysis showed that the rs4705341 was related to differentiated status, clinical stage I-II, and patients without lymph node metastasis. Moreover, patients with rs4705341GG had a longer overall survival . These findings indicate that the miR-143/145 rs4705341 may be used as a potential biomarker for the development and prognosis of CRC.The aim of this study was to investigate the effect of a polymorphism rs4705341 in the flanking region of miR-143/145 on the risk of colorectal cancer (CRC). The rs4705341 polymorphism was analyzed in 1002 cases and 1062 controls using a polymerase chain reaction-restriction fragment length polymorphism method. We found a significantly reduced CRC susceptibility with miR-143/145 rs4705341 in homozygote comparison (adjusted OR = 0.66, 95%CI, 0.50-0.88,  Colorectal cancer (CRC) takes almost 700,000 lives every year, making it one of the most common forms of neoplasia in industrial countries . As the MicroRNAs (miRNAs) are 19-25-nucleotides non-coding RNA molecules that can result in the degradation or repression of mRNA by sequence-specific base pairing on the 3′-untranslated regions of target mRNA , 7. ReceWe conducted a large-scale case-control study including 1002 CRC patients and 1062 healthy controls. The mean age of cases and controls was 60.7 and 57.9 years, respectively. The male/female ratio was about 3:2 in both case and control group. Among the cases, 564 (56.3%) were well-moderately differentiated CRC and 438 (43.7%) were poorly differentiated CRC. Five hundred eighty-four (58.3%) were stage I–II, and 418 (41.7%) were stage III–IV. Most of the cases (64.7%) had no lymph node metastasis (Table P = 0.09 and 0.22), indicating that we obtained representative samples of the general population. As shown in Table P = 0.004; G vs. A: adjusted OR = 0.83, 95%CI, 0.73-0.94, P = 0.004). Similarly reduced risk was also observed in a dominant model  and recessive model .The distribution of the rs4705341 polymorphism was consistent with the Hardy-Weinberg equilibrium in the case and control group  or poorly-undifferentiated CRC patients . The protective effect was also found in patients with clinical stage I-II  but not in patients with clinical stage III-IV. Moreover, the frequencies of GG genotype and G allele were significantly different in patients without lymph node metastasis  rather than in patients with lymph node metastasis .Log-rank test and Cox proportional hazards model were performed to examine the association of the rs4705341 with 3-year overall survival of CRC. The mean survival time in AA carriers was 22.7 months, whereas the mean survival time in AG/GG carriers was 26.6 months. Significant difference was observed between the 2 groups with a In this study, we confirmed an association of the rs4705341 polymorphism in the flanking region of miR-143/145 with the risk of CRC in a Chinese population, with the rs4705341G allele having a 0.83-fold decreased risk of CRC. Stratification analysis revealed that the rs4705341 was related to clinical features of CRC patients. These findings indicate that the rs4705341 polymorphism may be involved in the etiology and prognosis of CRC.Recently, genetic variants in the flanking region of miRNA may contribute to individual's susceptibility to cancer –24. An rSeveral limitations should be discussed. CRC is a complex disease with multi-factors rather than a single factor involved. In this study, only genetic factor was taken into consideration. Further association studies investigating gene-gene and gene-environment interaction are of great importance to clarify the pathogenesis of CRC. Distribution of the rs4705341 is different in diverse ethnic groups. Only Chinese Han subjects were enrolled in this study. Therefore, the results cannot be directly extended to other ethnicities. Further investigations are warranted to validate the results in different races. Although we found that the rs4705341 polymorphism is potentially functional, the convincing mechanism is not fully provided. Further analysis of exact mechanism is needed.In conclusion, we reported for the first time that the rs4705341 GG genotype was associated with a decreased CRC susceptibility and better outcome, suggesting that the miR-143/145 rs4705341 may be a potential biomarker for the development and prognosis of CRC.CRC patients were consecutively recruited from the West China Hospital of Sichuan University, the Luoyang Central Hospital Affiliated to Zhengzhou University, and the Affiliated Hospital of North Sichuan Medical College. Eight hundred and sixty-five paraffin-embedded tissue specimens (10 μm thickness) and 256 peripheral venous blood samples from CRC patients were collected between January 2010 and February 2015. The diagnosis of CRC was determined by histopathological analysis. Clinical information, including age, gender, differentiated status, clinical stage and lymph node metastasis, was retrieved from surgical and pathological records and summarized in Table Genomic DNA from EDTA-anticogulated peripheral blood was extracted by using a commercial extraction kit  and DNA from paraffin-embedded tissue sections was isolated by using a Puregene® DNA isolated kit  according to the instruction manual.Bcl I . The rs4705341A allele was cut into two fragments of 106 and 17 bp, whereas the rs4705341G allele remained intact. For quality control, the laboratory staff performing the genotyping analysis did not know the subjects' case-control status. Two research personnel independently read the gel pictures. The samples were reanalyzed if an agreement was not reached. Moreover, the genotyping method of PCR-RFLP was confirmed by sequencing  in 10% of all samples, and the results were all consistent. Due to DNA quality and quantity, 1062 controls and 1002 cases were genotyped successfully.The rs4705341 polymorphism was analyzed using a polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The primer sequences were 5′-CCTCGGCTTCCCAAAGTGATC-3′ (forward) and 5′-TCCCAGCCAGATAACTGCTTTCC-3′ (reverse). PCR reaction was performed in a total volume of 10 μl, containing 50 ng genomic DNA, 20 pM each primer, and 5 μl 2X PCR mix . The PCR products were digested with 2 test. The χ2 test, with odds ratio (OR) and respective 95% confidence interval (CI), were applied to evaluate the differences of genotype and allele distribution among cases and controls. Genotypic association tests were determined using SNPstats [P level of < 0.05 was considered as statistically significant.All the analyses were done using SPSS software version 13.0 . Hardy-Weinberg equilibrium was measured by χSNPstats , assumin


Overgrazing is a severe problem in several regions in Northwestern China and has caused serious land degradation. Secondary natural succession plays an important role in the accumulation of soil carbon and nitrogen contents. Estimating the effects of grazing exclusion on soil quality and plant diversity will improve our understanding of the succession process after overgrazing and promote judicious management of degraded pastures. This experiment was designed to measure soil properties and plant diversity following an age chronosequence of grasslands  in Northwestern China. The results showed that continuous fencing resulted in a considerable increase in plant coverage, plant biomass (above- and below-ground biomass), and plant diversity, which can directly or indirectly improve the accumulation of soil organic carbon and total nitrogen content. The plant coverage and the above- and below-ground biomass linearly increased along the succession time, whereas soil organic C and N contents showed a significant decline in the first 12 years and, subsequently, a significant increase. The increased plant biomass caused an increase in soil organic carbon and soil total nitrogen. These results suggested that soil restoration and plant cover were an incongruous process. Generally, soil restoration is a slow process and falls behind vegetation recovery after grazing exclusion. Although the accumulation of soil C and N stocks needed a long term, vegetation restoration was a considerable option for the degraded grassland due to the significant increase of plant biomass, diversity, and soil C and N stocks. Therefore, fencing with natural succession should be considered in the design of future degraded pastures. Overgrazing has accelerated soil erosion and severe degradation in arid and semiarid grasslands over recent decades ,2,3 as iThe installation of fencing and vegetation restoration have been implemented to reduce the risks of serious soil erosion and reverse degradation trends ,8,10. FoGrazing exclusion through fencing is regarded as the most effective method available for ecological restoration on the Loess Plateau of China because of its complex terrain, extreme drought conditions, and severe soil erosion . AlthougPrevious study in the Yunwu Mountain reserve showed that soil bacterial communities had a successional dynamic along a chronosequence . AlthougStipa bungeana, Thymus mongolicus, and Artemisia vestita [The experimental site, the Yunwu Mountain Reserve, is located in Guyua City, Northwest China, and is a typical grassland with different successional ages after fencing. In this study, we chose four successional ages, including the one-year site, 12-year site, 20-year site, and 30-year site. Prior to fencing, the grassland was seriously degraded due to overgrazing. The soil type of the experimental site was entisols based on the American taxonomy  ,14,15. T2 quadrants were randomly established across each plot to represent heterogeneity. The biomass was estimated by collecting the above-ground biomass (AGB) and below-ground biomass  of all plants in one 1-m2 sampling quadrants in each plot. We dipped the 0–40 cm soil layer and sieved to obtain the roots (BGB). All roots were washed with distilled water, air-dried, and then oven-dried at 70 °C for at least 72 h or more to a constant weight before weighing. The total biomass (TB) was the sum of the AGB and BGB. TB, AGB and BGB were expressed as t/ha.Soil samples were obtained during mid-August 2014 when the biomass had reached its peak for the successional sites. For each successional age, we selected five sampling sites as replicates for a total of 20 sampling sites (4 ages × 5 replicates) . The domS). The Shannon-Wiener Index was calculated using the Equation (1) [iP is the relative abundance of each species; S is the total number of species; and ln is the log base-e. The number of plant species was used to estimate the species richness (tion (1) ,17:(1)H=Soil samples were collected using seven soil cores in each plot. Soil core was obtained using a 5-cm diameter soil auger and pooled to a composite sample. The soil samples were obtained from 0 to 20 cm. The roots, stones, and animals were removed, air-dried, and finely ground through a 0.15-mm sieve for the C, N and P analysis.3 ring (three replicates in each sample site) [contentelement is the soil organic carbon content (%), total nitrogen (%) and total phosphorus (%), respectively; BD is the soil bulk density ; and d is the soil depth (cm) of 20 cm.Soil bulk density (BD) was measured using the undisturbed core method, using a 100 cmle site) . The conle site) . The totle site) ,21:(2)ElAll the data in the present study were expressed with mean ± standard error. The one-way analysis of variance (ANOVA) was analyzed to investigate the effects of successional age on the soil properties studied and the plant characteristics. The Pearson correlations or linear regressions were used to determine the relationships between the nutrients in soil and plant characteristics. The principal component analysis (PCA) was conducted using Canoco 5.0  for Windows to test the relationships between soil C, N, and P content and plant characteristics. ANOVA and Pearson correlation analyses were conducted using the SPSS 20.0 software . The significance was at the 0.05 level. The figures were performed using Origin Pro 2016 .p < 0.05). As expected, plant TB, AGB, and BGB significantly increased with successional age after fencing. Plant AGB increased from 0.19 to 0.85 t/ha, with the highest result obtained in the 20-year site (0.93 t/ha), but there was no significant difference between the 20-year and the 30-year site (p > 0.05). Plant BGB increased in the first 20 years, then decreased with fencing time, and reached a peak at the 20-year site (0.51 t/ha). TB showed a similar trend with BGB, with a significant lower result in the one-year and 12-year sites than the 20-year and 30-year sites (p < 0.05). Plant cover significantly linearly increased from 31% to 90% (p < 0.05), reaching a peak at the 30-year site (90%). There was a significant difference across all succession stages (p < 0.05). The H index could indicate the diversity of the plant community in the grassland with a significant higher H index in the 20-year and the 30-year sites than those in the one-year and 12-year sites (p < 0.05). The S index also showed similar varied trends with the H and Shannon indices, with a range from 13 to 26. The H index and S index remained stable after a 20-year succession, whereas plant cover and AGB continued to increase. H and S showed no significant difference between the one-year and the 12-year sites, as well as between the 20-year and the 30-year sites (p > 0.05).In this study, the plant diversity indices and plant biomasses showed some remarkable successional changes along a chronosequence , suggestp < 0.05) (p < 0.05). In the first 12 years, STN contents decreased by 34%, then increased 51% after 30 years. The range of STP concentration was rather narrow (0.62–0.72 g/kg), with no significant difference among the restoration times (p > 0.05) (The vegetation restoration age had significant effects on the nutrient contents in soil. The concentration of SOC showed a significant decline in the first 12 years and then significantly increased with a peak at the 30-year site ( < 0.05) . The pat < 0.05) . STN con > 0.05) .−3 and was significantly lower at the 20-year and 30-year sites (p < 0.05) (p < 0.05). There were no significant differences between the one-year and the 12-year sites, and the 20-year and the 30-year sites (p > 0.05) (Soil bulk density ranged from 0.94 to 1.15 g·cm < 0.05) . Bulk de > 0.05) .SOC stocks ranged between 24–40 t/ha and showed a similar pattern as the SOC contents, but were less explicit . Soil N p < 0.05) (p < 0.05) , TN contents , SOC stocks , TN stocks , AGB , and BGB  ( < 0.05) . There w < 0.05) . These r = 69.7) .p < 0.05). The lowest SOC content and stock were found for the 12-year site, which can be explained by the fast growth of plants during the early successional period. The restoration for soil nutrients is a slow process and it needs more time. Significantly higher SOC contents were found for the 20-year site (p < 0.05), which was confirmed by the previous study [Medicago in China (0.27 t/ha per year) [The results showed that the vegetation succession with fencing significantly influenced most of the studied soil properties. A significant increase occurred in the 20-year site  .p < 0.05). Similar to the results found for SOC, the lowest TN contents were found at the 12-year site. Significantly higher contents were found for the 20-year and 30-year sites (p < 0.05), which was in accordance with previous results [Vegetation succession also significantly influenced the soil TN ( results ,23. In t3 to 0.94 g/cm3. Similar variations were reported in the Loess Plateau [Along with the succession, the soil bulk density decreased from 1.15 g/cm Plateau  and the  Plateau . A signi Plateau ,28. The  Plateau .Our study also demonstrated that plant biomass had a significant effect on the soil properties studied . Long-tePlant diversity strongly influences ecosystem functions and services, such as soil carbon and nitrogen storage. Plant species richness and the Shannon index enhanced SOC and TN stocks, which have been confirmed by previous studies ,38. In tOn the other hand, fencing can also enhance the ability to resist soil erosion via plant coverage . The incOvergrazing is one of the main causes of land degradation in the semi-arid grasslands of Northwestern China. Continuous fencing resulted in a considerable increase in plant coverage and biomass, which enhanced the resistance to the loss of soil organic C and N and subsequently caused the accumulation of soil organic carbon and total nitrogen. These results indicated that vegetation restoration with fencing on degraded grasslands has great potential to improve soil fertility, sequester soil organic carbon and nitrogen, and improve plant above- and below-ground biomass. Soil and plant restoration was an incongruous process. Soil restoration was a slow process and fell behind vegetation restoration after long-term fencing. Compared with the above-ground biomass, the below-ground biomass was the main driver behind the accumulation of SOC and TN contents. Therefore, fencing with natural restoration should be considered in the management of degraded pastures in the Loess Plateau.


This study aimed to investigate the possible effect of omega-3 fatty acids on reducing the mortality of sepsis and sepsis-induced acute respiratory distress syndrome (ARDS) in adults.Medline, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI) database, WangFang database, and Chinese BioMedical Literature Database from their inception to March 6, 2017, were searched using systematic review researching methods. Five factors were analyzed to investigate the correlation between omega-3 fatty acids  and mortality rate.p = 0.39). But in EN subgroup, omega-3 fatty acids treatment seemed to have some benefits in reducing mortality rate (p = 0.04). In the RCTs including similar baseline patients, partial correlation analysis found that the concentration ratio of n-6 to n-3 fatty acids had positive correlation with reduction of mortality (RM) , whereas the total number of each RCT had negative correlation with RM .Forty randomized controlled trials (RCTs) were initially included, but only 25 of them assessed mortality. Of these RCTs, nine used enteral nutrition (EN) and 16 used parenteral nutrition (PN). The total mortality rate in the omega-3 fatty acid group was lower than that in the control group. However, the odds ratio (OR) value was not significantly different in the EN or PN subgroup. Eighteen RCTs including 1790 patients with similar severity of sepsis and ARDS were also analyzed. The OR value was not significantly different in the EN or PN subgroup. Omega-3 fatty acids did not show positive effect on improving mortality of sepsis-induced ARDS (This review found that omega-3 fatty acid supplementation could reduce the mortality rate of sepsis and sepsis-induced ARDS. However, further investigation based on suitable concentrations and indications is needed to support the findings. Sepsis is a critical infectious condition that can cause immune system response and organ dysfunction . AntibioThe immune response is supposed to be correlated with sepsis . Two phan-6 polyunsaturated fatty acids (PUFA) of the inflammatory cell membrane with n-3 PUFA, reduces the production of arachidic acid from n-6 PUFA, protects the fluidity of CD4+ lymphocyte membrane, and stabilizes the cell membrane , concentration ratio of The searches found 2114 records after duplicates were removed. Of these, 2066 papers were excluded because they were reviews, nonhuman research, controlled studies, or RCTs comparing different ablative methods. Of the remaining 48 full texts that were selected initially and read through, 40 RCTs were initially included  II score, and one reported the baseline APACHE III score. Eight RCTs reported the Sepsis-related organ failure assessment (SOFA) score, and three RCTs reported the baseline Marshall score , and other 13 studies compared soybean oil plus omega-3 fatty acids to soybean oil alone (A + B vs B). All characteristics of studies are shown in Table A total of 25 RCTs were included in mortality analysis Fig. .Fig. 3CoI2 value was 0% in the EN group and 40% in the PN group. A fixed-effects model was used for meta-analysis. The P value was 0.12 and 0.20, respectively, which demonstrated that omega-3 fatty acids had no significant effect on reducing the mortality of sepsis and ARDS.Nine RCTs with 1128 patients compared different types of EN with various concentrations of omega-3 fatty acids for treating sepsis and ARDS. Further, 126 patients in the omega-3 fatty acid group (22.18%) and 144 patients in the control group (25.71%) died at the end of the observation period. Another 16 RCTs including 1289 patients compared PN with the conventional treatment of sepsis. Moreover, 160 patients in the omega-3 fatty acid group (accounted for 24.62%) and 178 patients in the control group (27.86%) died at the end of observation period. The heterogeneity among studies in each subgroup (EN and PN) was low. The Data from 18 RCTs including 1790 patients with severe sepsis and sepsis-induced ARDS having similar severities were analyzed Table , Fig. 3bI2 = 27%), and mortality was insignificantly low in the high-concentration omega-3 fatty acid subgroup .Five RCTs with 722 patients were included in the EN subgroup. Further, 83 patients died in the high- and low-concentration omega-3 fatty acid subgroups, respectively. The heterogeneity among studies was low (P = 0.32).Thirteen RCTs with 1068 patients were included in PN subgroup. The heterogeneity among studies was medium (50%). A fixed-effects model was used for analysis. Moreover, 132 patients in the omega-3 fatty acid group (24.54%) and 145 patients in the control group (27.36%) died at the end of the observation period (p = 0.39). But in EN subgroup, omega-3 fatty acids treatment seemed to have some benefits in reducing mortality rate (p = 0.04). The results were seen in Fig. Eight RCTs including 927 patients with sepsis-induced ARDS. Among these RCTs, six trials clearly indicated that they included patients with ARDS –34. In an-6 to omega-3 fatty acids, treatment duration, methods of nutrition support, comparison methods, and TN, had no correlation with RM. However, when data from RCTs including patients with similar severity were analyzed, the concentration ratio of n-6 to omega-3 fatty acids was found to have a positive correlation with RM , whereas the total number of each RCT had a negative correlation with RM . Other factors had no correlation with RM  at the end of study duration. Omega-3 fatty acids seemed to have no effect on reducing mortality when data from all these studies were combined. It was believed that severities affected the final result. However, the difference in mortalities between the treatment and control groups was also insignificant when data only from RCTs including severe sepsis and ARDS with similar severity were analysed. Although EN seems to reduce the mortality of sepsis-induced ARDS patients, the results only come from four small studies. Generally speaking, omega-3 fatty acids have no significant effect on sepsis-induced ARDS. Furthermore, concentration ratio of Staphylococcus-induced sepsis [n-3 polyunsaturated fatty acids had an antioxidant effect, they might worsen septic shock–induced vascular dysfunction, and not improve the survival rate of the disease [Animal experiments demonstrated the anti-inflammatory effect of omega-3 fatty acids in sepsis . A cecald sepsis . Some re disease . The res disease –23. Howe disease . Althoug disease . Inflamm disease . TherefoSome factors such as nutrition supplementation, concentration ratio n-6 to n-3 fatty acids, and interventional comparison may affect the impact of omega-3 fatty acids in treating sepsis or ARDS. Omega-3 fatty acids can be applied through enteral and parenteral routes. Recently, PN has been shown to be insufficient in improving the prognosis of critically ill patients . A studyn-6 to omega-3 fatty acids, the more the RM. On the one hand, this result implied that the ratio was not the higher the better. On the other hand, it has been found that the concentration ratio of n-6 to omega-3 fatty acids in the RCTs is equipped with a certain standard. It should be noted that omega-3 fatty acids alone may not play an anti-inflammatory role. Therefore, a comparison of n-6 and omega-3 fatty acids alone may not make sense.A certain proportion of n-3 and n-6 fatty acids is specified to be used for anti-inflammatory effect. Previous researches suggested that 1:2 –1:4 would be a better proportion for eliminating the inflammatory reaction . The preResearch methodologies of most studies are suitable. However, still some RCTs have a high risk of bias. The most common bias is allocation concealment. Half of these studies were single-blind trials. However, how this bias affects the mortality is still unknown. One of the interesting results is that the total number of studies has a negative correlation with RM. It means that the larger the scale of the study, the less the RM will be. The estimation of sample size in clinical trials is one of the most important issues in the research design. In general, the larger the research scale, the closer the result. On the contrary, an increase in the number of subjects may lead to more wastage of resources . Almost Although omega 3 fatty acid is a kind of fat preparation, no evidence demonstrates that it could increase blood lipid. On the contrary, studies suggest that fish oil can reduce serum triglyceride (TG) , and theOmega-3 fatty acid supplementation could reduce the mortality rate of sepsis and sepsis-induced ARDS, but this finding is still not supported by most evidence. Hence, investigating based on suitable concentrations and indications may be the directions of future research.


Caligus rogercresseyi, present on farmed Atlantic salmon  in Chile. The genome encodes the five canonical rhabdovirus proteins in addition to an unknown protein, in the order N-P-M-U (unknown)-G-L. Phylogenetic analysis showed that the virus clusters with two rhabdoviruses  obtained from another parasitic caligid, Lepeophtheirus salmonis, present on farmed Atlantic salmon on the west coast of Norway.We have determined the complete genome sequence of a new rhabdovirus, tentatively named Caligus rogercresseyi rhabdovirus Ch01 (CrRV-Ch01), which was found in the parasite The online version of this article (10.1007/s00705-018-3768-z) contains supplementary material, which is available to authorized users. C. rogercresseyi (sea louse) is a serious problem for farming of Atlantic salmon in Chile [C. rogercresseyi, but sequences of possible rhabdovirus origin  have been detected in the parasite. However, these sequences have been suggested to be possible endogenous viral elements (EVEs), i.e., viruses integrated in the germline genome of their hosts and inherited vertically [L. salmonis [C. rogercresseyi using EVEs/IREs available in the GenBank database (NCBI) as a starting point.The parasitic copepod in Chile –3. Therertically –14, or irtically . This wasalmonis , but Øklsalmonis  showed tC. rogercresseyi (taxid: 217165). Additional sequences where identified using BioEdit [C. rogercresseyi transcriptome shotgun assembly (PRJNA234316). A rhabdovirus genome sequence  based on three sequences  was assembled using Contig Express from the Vector NTI® Suite 9.0.0 (Thermo Fisher).Sequences resembling partial rhabdovirus genomes were identified by BLAST search  using se taxid: 2165. AddiC. rogercresseyi, using the assembled genome sequence as the basis for primer design. The lice were collected from the skin of farmed Atlantic salmon at three farming sites in Los Lagos (Region X) on the coast of Chile in 2016. The three farming sites are located with an average distance of 65 km. The parasites were fixed in RNAlater® (Thermo Fisher) and transported to the University of Bergen, Norway. RNA was extracted from the lice using TRI Reagent (Sigma-Aldrich), with the following modifications of the standard protocol: The lice were homogenized in 1 ml TRI Reagent with a 5-mm bead for 7 min at 50 Hz in a Tissuelyser LT (QIAGEN), and the RNA pellet was washed with an additional 1 ml of 100% ethanol before air-drying.The complete genome sequence  of the new rhabdovirus, named CrRV-Ch01 (accession no: KY203909), was obtained by Sanger sequencing of RNA from RNA from 4-5 pooled lice from one location was ligated to allow circularization and sequencing of the genome termini of CrRV-Ch01. To increase the efficiency of RNA ligation, the 5’-triphosphate residues of the RNA were removed by incubating 5 μg of total RNA with 5 units of 5’ RNA pyrophosphohydrolase  in 40 μl of 1X NEBuffer 2 for 30 min at 37 °C , followeThe resulting complete virus genome sequence of CrRV-Ch01 differed by 1.1% from the assembled sequence in the GenBank database and contained six open reading frames (ORFs) that were at least 408 nucleotides (136 amino acids) in length, in the order 3’-N-P-M-U-G-L-5’, i.e., different from the standard five-gene arrangement found in many of the rhabdoviruses. The leader region of CrRV-Ch01 is 78 nucleotides long, while the trailer region is 67 nucleotides long. The first 21 nucleotides of the leader region and the last 21 nucleotides of the trailer region exhibit an inverse complementarity of 81.8%. The putative transcription termination/polyadenylation signal (TTP), based on its homology to other rhabdoviruses, is conserved in the genome of CrRV-Ch01 and comprises the motif TATG(A)7. The transcription initiation (TI) sequence, AACAA, is present for all genes except for the U protein gene, which has AATAA as its TI sequence.C. rogercresseyi in the Pacific Ocean (Chile). Further amino acid sequence comparisons with other rhabdoviruses using BLAST search showed that CrRV-Ch01 shares 27.7% identity and 48.0% similarity with the virus Lepeophtheirus salmonis rhabdovirus No127 (LSRV-No127) present in L. salmonis in the North Atlantic, while the amino acid sequence similarity to both Bivens Arm virus and Tibrogargan virus (genus Tibrovirus) is 40.0%. The N protein contains the sequence 283QLSTRSPYST292, which shares high similarity with the RNA-binding motif (G(L/I)SXKSPYSS) present in vesiculoviruses, ephemeroviruses and lyssaviruses [The putative nucleoprotein gene (N gene) in CrRV-Ch01 is 1425 nt long and contains a single ORF of 1356 nt encoding a putative protein of 452 amino acids (accession number: APF32073). The putative N protein of CrRV-Ch01 shows 98.3% nucleotide sequence identity (98.0% amino acid sequence similarity) to a partial putative rhabdovirus nucleoprotein sequence  obtained from aviruses .The putative phosphoprotein gene (P gene) of CrRV-Ch01 is 807 nt long and contains a single ORF of 759 nt encoding a putative protein of 253 aa (accession number: APF32074). It contains 35 potential serine/threonine phosphorylation sites and five tyrosine phosphorylation sites. This putative P protein sequence shares no significant sequence identity to any sequences in the GenBank database and its identity is only suggested based on its size and genome position.14PVASAPTVPGP29.The putative matrix protein gene (M gene) in CrRV-Ch01 is 739 nt long and contains a single ORF of 687 nt encoding a putative protein of 229 aa (accession number: APF32075). Amino acid sequence comparison with other rhabdoviruses showed that it shares 24.6% and 25.1% identity and 42.4% and 40.2% similarity with the matrix protein genes of the salmon louse rhabdoviruses LSRV-No127 (AIY25914) and Lepeophtheirus salmonis rhabdovirus No9 (LSRV-No9) (AIY25909), respectively, but no significant match with other rhabdoviruses. A potential domain, PPPY/P(S/T)AP, which is necessary for efficient budding of the virus [The putative unknown protein gene (U gene) in CrRV-Ch01 is 439 nt long with a single ORF of 408 nt encoding a putative protein of 136 aa (accession number: APF32076). The U gene nucleotide sequence and the putative protein sequence share no detectable sequence similarity with any other viruses in the public databases. However, topology analysis of the putative U protein using the Phobius server predict an N-terminal signal peptide from aa 1 to 19 and an ectodomain from aa 20 to 136, but no transmembrane region. Additional protein coding genes have been described previously in several rhabdovirus genomes, including genes inserted between the M and G genes –29. They140NDSD, 242NKTS, 368NKSS, 420NDSS, and 476NGTS, respectively. BLAST searches showed that CrRV-Ch01 shares the highest amino acid sequence identity (22.3%) with a virus detected in a salmon louse , LSRV-No127 . The amino acid sequence identity to the classified anguillid perhabdovirus (accession no. AIY29111) is 20.7%.The glycoprotein gene (G gene) in CrRV-Ch01 is 1647 nt long and contains a single ORF of 1611 nt encoding a putative protein of 537 amino acids (accession number: APF32077). Topology analysis using the Phobius server predicted an N-terminal signal peptide (aa 1–17), an ectodomain from aa 18 to 493, transmembrane region spanning from aa 494 to 516, and a C-terminal cytoplasmic tail from aa 517 to 537. The protein is predicted to contain five putative N-glycosylation sites, Rhabdoviridae, with the closest relationships to the L proteins of LSRV-No9 (44.2% identity) and LSRV-No127 (44.0% identity). The vesiculoviruses Maraba virus, Cocal virus, Perinet virus, and Indiana virus are slightly more distant, showing 38.3, 38.3, 38.6, and 38.5% identity, respectively. Six blocks of conserved sequences are shared among the L proteins of members of the family Rhabdoviridae [The L gene of CrRV-Ch01 is 6429 nt long and contains a single ORF of 6390 nt encoding a putative protein of 2130 aa (accession no. APF32078). The L protein gene shows a clear affinity to those of other members of the family Rhabdoviridae were aligned using MAFFT. Ambiguously aligned regions in the alignment were removed using TrimAl [L. salmonis) present on salmon in the North Atlantic. The phylogeny of these rhabdoviruses from copepods shows no clear affinity to members of any of the known rhabdovirus genera.To investigate the relationship of CrRV-Ch01 to other members of the family Rhabdoviridae, a phylogenetic tree was generated based on the L protein  from C. rogercresseyi (Table C. rogercresseyi (Crog-18S) was used as internal control. The real-time RT-PCR reaction was run in a 12.5-μl reaction mixture containing 6.25 μl of 2X RT-PCR, 1.0 μl of 10 mM forward primer, 1.0 of μl 10 mM reverse primer, 0.22 μl of 10 mM probe, 0.25 μl of 25X RT-PCR enzyme mix, 1.75 μl of RNase-free water and 2.0 μl of template. The real-time RT-PCR analysis was run using an Applied Biosystems 7500 Fast Real-Time PCR System under the following conditions: reverse transcription at 45°C for 10 min, polymerase activation at 95°C for 10 min, and 45 cycles of DNA dissociation at 95°C for 15 and annealing/elongation at 60°C for 45 s. The virus was present in both male and female lice from all three locations in Chile. The prevalence of positive lice was 72 % (36 of 50 lice) and ranged from 64.2 to 85.7% for the three locations. The CT value of positive lice ranged from 12.6 to 35.8 and the average CT value was 27.7, ranging from 25.2 to 29.9 for the three locations. Four individuals tested strongly positive (Ct < 15) for the virus. The internal control assay, Crog-18S, gave Ct values ranging from 4.3 to 10.0  showing the presence of RNA in all samples. Viral RNA was also detected in egg strings from three positive female lice . The presence of viral RNA in egg strings could indicate, as described for the L. salmonis rhabdoviruses [To estimate the prevalence of CrRV-Ch01 in adult oviruses , that veCaligus rogercresseyi, CrRV-Ch01, collected from farmed Atlantic salmon in Chile. CrRV-Ch01 was not present in all the specimens of C. rogercresseyi tested, and we have found what appears to be a complete, functional genome. The completeness of the genome sequence, the presence of complete genome termini exhibiting inverse complementarity, TI and TTP sequences for all genes, and ORFs for all essential rhabdovirus proteins, makes it highly unlikely that this virus is integrated into its host genome. To yield the results of this study, an endogenous virus genome would have to be transcribed as a complete negative-sense viral genome with no post-transcriptional modifications. Thus, this study strongly suggests that CrRV-Ch01 is indeed an exogenous virus rather than another example of endogenous viral elements [in situ hybridization, and electron microscopy to examine the pathology and tropism of the virus and presence of viral particles. The presence of CrRV-Ch01 in Atlantic salmon has not been studied. Because the lice feed on the mucus, skin and blood of their host, it is possible that we have detected a salmonid virus in the gut of the lice. However, the presence of the CrRV-Ch01 genome in the egg strings of the lice strongly suggests otherwise. Nevertheless, future studies should investigate the presence of CrRV-Ch01 in Atlantic salmon and other fish frequently infested with C. rogercresseyi. This could reveal if this is an arbovirus infecting both fish and lice, or solely a caligid virus. The closely related LsRV-No9 and LsRV-No127 have been detected in the skin of Atlantic salmon at the attachment site of chalimi, but there are no evidence of infection or replication [To summarize, we have characterized the first rhabdovirus genome from the parasitic copepod elements . To supplication .Caligidae-specific association of this virus clade that has probably existed for a considerable time. This indicates that there may be several undiscovered rhabdoviruses infecting other members of the family Caligidae.The phylogenetic clustering of CrRV-Ch01 from Chile with the rhabdoviruses LsRV-No9 and LsRV-No127 from salmon lice in the North Atlantic, i.e., the demonstration of the presence of these related viruses over a large geographical area, suggest a Supplementary material 1 (PDF 30 kb)Below is the link to the electronic supplementary material.


The aim of this study was to investigate the correlations between serum cartilage oligomeric matrix protein (COMP), interleukin-16 (IL-16) and different grades of knee osteoarthritis (KOA) in Malaysian subjects.Ninety subjects were recruited comprising 30 with Kellgren-Lawrence (K-L) grade 2 KOA, 27 with K-L grade 3 KOA, 7 with grade 4 KOA, and 30 healthy controls. All subjects completed the Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire. Serum COMP and IL-16 levels were measured using ELISA and their values log transformed to ensure a normal distribution.p = 0.001) and its subscales, pain (p = 0.005), stiffness (p = 0.019) and physical function (p<0.0001). Serum IL-16 levels were significantly higher in Malaysian Indians compared to Malays and Chinese (p = 0.024).There was no significant differences in levels of log serum COMP and IL-16 between healthy controls and KOA patients. There were no significant differences in the log serum COMP and IL-16 levels within the different K-L grades in the KOA patients. In KOA patients, log serum IL-16 levels significantly correlated with the WOMAC score (In this multi-ethnic Malaysian population, there was no difference in serum COMP and IL-16 levels between healthy controls and patients with KOA, nor was there any difference in serum COMP or IL-16 levels across the various K-L grades of KOA. However, there were significant inter-racial differences in serum IL-16 levels. Osteoarthritis (OA) is a “progressive disease of synovial joints that represents failed repair of joint damage that results from stresses that may be initiated by an abnormality in any of the synovial joint tissues, including articular cartilage, subchondral bone, ……… or synovium, which ultimately results in the breakdown of cartilage and bone” . It is tAlthough cartilage loss in OA is attributed to a mechanical failure of repair , recent For many years, OA assessment has relied on using plain radiograph scoring systems of which the Kellgren-Lawrence (K-L) grading system is the most well-known . HoweverSerum cartilage oligomeric matrix protein (COMP) is a non-collagen biomarker for cartilage degradation. Based on the results of a systematic review and meta-analysis, serum COMP was found to be consistently elevated in patients with radiographic knee OA (KOA) compared to healthy controls. In addition, higher levels of serum COMP are associated with a trend toward greater radiographic OA severity when compared to controls .Pro-inflammatory cytokines have been implicated in the pathophysiology of OA, including IL-1β, TNF, IL-6, IL-15, IL-17, IL-18, IL-21 and IL-8 . IL-16 wThere have been no previous studies of serum biomarkers in Malaysian patients with KOA. We therefore set out to study serum COMP, a well-established marker in OA, in our multi-ethnic population to assess if its level was different in KOA patients compared to healthy controls, and if there was any difference in levels with KOA disease severity. In addition, we performed an exploratory study measuring serum IL-16, a novel pro-inflammatory mediator, in the same population, with a view to trying to replicate the other 2 studies as well as getting information on IL-16 levels in a multi-ethnic population.Between November 2015 and March 2016, patients with primary KOA, over the age of 50 years, from the Orthopaedic outpatient clinics at Hospital Tuanku Ja’afar, Seremban, and Mawar Hospital, Seremban, were invited to participate in this study. Inclusion criteria were that patients had KOA as per the American College of Rheumatology clinical classification criteria using history and physical examination [All subjects completed the WOMAC (Western Ontario and McMaster Universities Arthritis Index) questionnaire; Version LK 3.1 English for Australia Index with the Likert scale was used. The WOMAC Index is a validated, self-administered questionnaire that is widely used in the evaluation of subjects with hip and knee osteoarthritis. It assesses pain, stiffness and physical function through a set of 24 questions . The hig2.5 mls of blood was withdrawn from each subject into a plain tube and allowed to clot at 4°C. The blood was then centrifuged for 15 minutes at 1,000 g, the supernatant serum was removed, aliquoted and stored at -20°C until use.Serum COMP was measured using the Human COMP Quantikine ELISA Kit (R&D Systems), a solid phase sandwich ELISA immunoassay. The assay range was 0.2–10 ng/mL. The intra-assay precision co-efficient of variation (CV) was 2.1–3.8% and the inter-assay precision CV was 4.3–4.8%.Serum IL-16 was measured using the Human IL-16 Quantikine ELISA Kit (R&D Systems), a solid phase sandwich ELISA immunoassay. The assay range was 31.2–2,000 pg/mL. The intra-assay precision CV was 4.1–5.1% and inter-assay precision CV was 11.5–12%. Both assays were performed according to the manufacturer’s instructions.http://www.openepi.com/) with confidence interval set at 95% and power at 80%. The results obtained were 28 in each group for serum COMP levels, and 16 in each group for serum IL-16 levels. For ease of recruitment, the numbers in each group was rounded up to 30 and recruitment stopped for each group stopped once this number was reached.The sample size was calculated using an online sample size calculator . The variables were analysed for skewness and as suggested by Bulmer , the datAll subjects gave signed informed consent prior to inclusion in the study. This study was approved by the Joint Committee of the Research and Ethics Committee of the International Medical University and was conducted in accordance in accordance with the ethical standards laid down in the 2013 Declaration of Helsinki.p = 0.011). There was no difference in the proportion of the different races  between the 2 groups (Pearson Chi-Square p = 0.125). The median age was significantly lower in the control group compared to those with KOA, 57.50 vs 62.50 years (p = 0.001) with an age range of 40 years. There was no significant differences between the 2 groups with regards to their body mass index (BMI), log serum COMP and log serum IL-16 levels. The WOMAC and its subscales scores were significantly higher in the KOA patients compared to healthy controls (p < 0.0001). The comparisons between the healthy controls and KOA patients as a group are shown in p = 0.097, p = 0.399 respectively).Ninety patients were recruited comprising 30 with K-L grade 2, 23 with K-L grade 3 and 7 with K-L grade 4 KOA, and 30 healthy controls. In the healthy control group there were 15 female and 15 male subjects. In the KOA group, there were 46 females and 14 males. There was significantly more females in the KOA group compared to controls (Pearson Chi-Square p = 0.044). In the WOMAC subscales, there were significant correlations between the pain (Spearman’s p = 0.023) and physical function (Spearman’s p = 0.048) but not the stiffness (Spearman’s p = 0.364) subscales. Log serum COMP significantly correlated with age (Spearman’s p = 0.005). There was no correlation between log serum COMP and IL-16 (Pearson correlation p = 0.111), even after correcting for age (Pearson correlation p = 0.122).There was a significant positive correlation between WOMAC and BMI . There was no significant differences in WOMAC and its subscales score, log serum COMP and log serum IL-16 levels between the 3 groups.The KOA patients were then divided into their K-L grade for analysis and the results are shown in p = 0.001). Log serum IL-16 also significantly correlated with the WOMAC subscales–p = 0.005 for pain, p = 0.019 for stiffness and p < 0.0001 for physical function. There was no significant correlations between log serum COMP, WOMAC scores or its subscales . Log serum COMP significantly correlated with age (Spearman’s p = 0.001).Pearson’s correlation tests was performed to determine the correlation between WOMAC score and its subscales, log serum COMP and log serum IL-16 levels in patients with KOA. Log serum IL-16 was significantly correlated with WOMAC  (p = 0.0p = 0.001). As shown in To our knowledge, this is the first study of serum biomarkers in KOA in a multi-ethnic Malaysian population. As cytokines are produced by various immune cells to modulate the cell-mediated and humoral response, they are more commonly studied in inflammatory conditions/inflammatory arthritides rather than in OA. The only other paper looking at cytokines in KOA published from Malaysia studied mRNA expression of IL-6 in KOA cartilage and found it no different from that in normal cartilage . In otheIn a systematic review and meta-analysis, serum COMP was found to be elevated in patients with radiographic knee OA compared to healthy controls and higher levels are associated with more severe disease . HoweverThe WOMAC questionnaire assess pain, stiffness and physical function in patients with OA. Overall, the WOMAC score was significantly higher in KOA patients compared to healthy controls which was not unexpected. However, the mean (anti-log) levels of IL-16 were not different in the 2 groups. In contrast, there was a strong correlation between the WOMAC score and serum IL-16 and the WOMAC score and BMI when directly compared. While there are no previous studies evaluating the correlation between serum IL-16 and WOMAC, it can be hypothesised that the positive correlation found in our study between the two variables could suggest that inflammation as measured by serum IL-16 influences pain and physical function as assessed by WOMAC. There have been other studies that have shown a link between WOMAC scores and the pro-inflammatory cytokines, IL-6 , IL-15  and TNFαThe positive correlation between WOMAC and BMI found in our study has been shown in previous studies. This is not unexpected as obesity is a well-known risk factor for OA . A higheOne of the limitations to this study is that the sample size was small, but a sample size calculation was done so the numbers were adequately powered to show a difference. When comparing between the healthy controls and KOA patients, the numbers should have been adequately powered to show a difference as the calculations showed that only 28 patients were required in each group for the COMP and 16 patients for IL-16. As we had 30 controls and 60 patients, we should have been able to show a difference between the controls and KOA patients but we did not. However, the numbers of patients with K-L grade 4 KOA was small which may have affected the results when comparing the biomarker values with KOA disease severity.We note that our control subjects had a positive WOMAC score. This may be because our control subjects are older, rather than younger. However, there have been 2 studies that have looked at the WOMAC score in a “normal” population. In the first study, the average score for physical function for both males and females was 1.78 . In the An interesting result of this study was to find that the serum IL-16 levels are different in the 3 major ethnic groups in Malaysia, and that the levels we have found are different from that found in Luo and colleagues’ study . We feelThis is the first study looking at serum biomarkers in Malaysian patients with KOA. Two biomarkers were studied–serum COMP which has been well-studied in different KOA populations but not so commonly in Asian populations, and serum IL-16, a recently suggested OA biomarker of which there has been only 2 prior publications. We found that there was no difference in serum COMP and serum IL-16 levels between healthy controls and patients with KOA, and no significant difference in levels of both biomarkers across the various K-L grades of KOA. There was a significant correlation between WOMAC and serum IL-16 which may suggest that serum IL-16 levels can correlate with symptoms. Of interest is that serum IL-16 levels are significantly different between the 3 major ethnic groups in Malaysia, which warrants further investigation.S1 File(PDF)Click here for additional data file.